Measurement of Sugars and Alcohol on HPLC

Materials and Equipment

• HPLC withRefractive Index Detector
  • Bio-Rad Aminex HPX-87H Column
  • Cation H deashing gaurd column (BioRad #125-0129) placed inside the column heating unit,
• 2 µm filters for syringe end   
• 1 ml syringe   
• HPLC vials
• 1ml Pipette with pipette tips

Reagents

Reagent	Concentration	Units	CAS Number
Sulfuric Acid Mobile Phase	0.005	Molar	7664-93-9

Procedure

HPLC Settings

Use the following settings for HPLC: Column: Injection volume: 50 µL, Flow rate: 0.6 ml min^-1, Column temperature: 65°C, Detector temperature: 30°C, Run time: 30 minutes. HPLC should be calibrated at 4, 2, 1, 0.5, and 0.25 g L^-1 with glucose.

Sample Preparation:

Solids should be removed to the extent possible with centrifugation and filtration. Samples should be able to pass through a 2um syringe without significant resistance.

1.      Centrifuge sample for 5min at 5000 rcf in centrifuge or microcentrifuge. 

2.      Pipette ~1.25 ml of decant into a 1.5ml microfuge tube taking care to avoid transferring solids. Place microfuge tubes in Microcentrifuge and centrifuge for 10min@ 16000 rcf.

3.      Pull a sample with a 1ml syringe from the microfuge tube and filter though a 0.2 µm filter into the HPLC vial.

4.      Run through HPLC using startup Wizard (The following is a generalized procedure for Waters Software)

a.       Select LC

b.      Select number of injections (depends on what is being analyzed)

c.       Injection volume is 50 (micro liters) and run time is 35 minutes, unless stated otherwise.

d.      Select Method and name samples

6.      Fill out the name of the HPLC run in the notebook

7.      Process HPLC results

Common Mistakes

Mistake	Evidence of the Mistake	Cause	Effect on Results